Solid Medium for the Storage of Biological Material

ABSTRACT

This invention relates to flat solid media for the storage of samples of biological materials and methods of analysing biomolecules contained within the samples following storage. In particular, the invention relates to the storage and further analysis of biomolecules present in the biological materials, such as proteins, enzymes and nucleic acids. The invention finds particular utility in the dry, room temperature storage of biological materials.

CROSS REFERENCES TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.13/798,483, filed Mar. 13, 2013, which claims priority benefit of GreatBritain Application No. 1301618.3 filed Jan. 30, 2013, each of which isincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to flat solid media for the storage of samples ofbiological materials and methods of analysing biomolecules containedwithin the samples following storage. In particular, the inventionrelates to the storage, recovery and further processing of biomoleculessuch as proteins, enzymes and nucleic acids.

BACKGROUND OF THE INVENTION

The use of solid media or supports such as filter paper for thecollection and analysis of biological materials such as human blooddates back to the early 1960s, when Dr. Robert Guthrie used dried bloodspot (DBS) specimens to measure the biomolecule phenylalanine innewborns for the detection of phenylketonuria (Mei, J., et al., 2001;Journal of Nutrition, 131:16315-1636S). This novel application forcollecting blood led to the population screening of newborns for thedetection of treatable, inherited metabolic diseases. DBS have now beenused for over 40 years to screen for a large range of neonatal metabolicdisorders including enzymes, proteins and for inherited disease usingnucleic acid analysis.

The gathering of biological materials such as DBS specimens is carriedout by spotting whole blood, for example, onto a solid support, such asa membrane, glass fiber or paper, either from venous blood or directlyfrom a finger or heel prick, making this method particularly suitablefor the shipment of specimens from peripheral clinics to centrallaboratories. Furthermore, DBS packed in zip-lock plastic bags withdesiccant can be stored and shipped at ambient temperature, thusavoiding the need for i) cold chain storage and ii) fast specializedtransportation. DBS collected by applying a drop of blood onto anabsorbent material such as Whatman 903 Neonatal STD paper are notsubject to the IATA Dangerous Goods Regulations (Addendum II, March2005).

Commonly, analysis of DBS is carried out for the presence of infectiousagents such as for the presence of human immunodeficiency virus (HIV) orother pathogens. Typically serological or nucleic acid tests are carriedout for this application.

The combination of DBS and the detection of endogenous proteinbiomolecules has been described in the scientific literature; forexample, the biomarker for cystic fibrosis (CF) is immunoreactivetrypsin (IT). The first reported use of endogenous IT from DBS for CFscreening was published by Ryley et al., in 1981 (J. Clin. Pathol. 34,906-910). Since then, it has been routinely used as an indicator of CFusing DBS from neonates. A number of commercial organisations supply FDAapproved immunoassay kits for this application. Many simply use a“paper-in” approach, in which a paper punch containing the DBS isapplied directly in to the immunoassay and the analyte of interest isextracted in situ. Recently (Lindau-Shepard & Pass, 2010, Clinical Chem.56, 445-450) demonstrated that IT exists in two different isoforms.These authors reported the development of a suspension (or paper-in)array-based immunoassay for the diagnosis of CF using the two differentisoforms of IT. All these protein-based studies were carried out onuncoated Guthrie cards (Whatman 903 paper).

Since the inception of anonymous human immuno-deficiency (HIV)screening, over 1.2 million DBS tests have been carried out for theserological detection of endogenous anti-HIV antibodies in the bloodfrom expectant mothers. These studies have proved that i) concerns aboutlong-term storage of blood and any associated proteins of interest haveproved unfounded and ii) the presence of haem in the DBS does notinterfere with assay performance.

Additional solid paper supports that are used for collecting,transportation and storing DBS and other bodily fluids for newborn andneonatal screening purposes include

-   -   1. Ahlstrom 226    -   2. Munktell TFN (CE marked)    -   3. Toyo Roshi grade 545 Advantec Toyo, Tokyo (see Elvers L et al        2007; J. Inherit Medtab Dis 30, 4, 609).

Slow desiccation or even a small degree of rehydration under conditionsof high relative humidity will allow the growth ofbiomolecule-destroying microflora. Even in the presence ofbacteriostatic agents of the type that do not denature proteins, therewill be conditions that permit enzymatic-autolytic breakdown of thebiomolecule and some non-enzymatic breakdown of the biomolecule (inenzymatic-autolytic breakdown, dying or damaged tissues, either humancells or parasite cells, activate enzymes that degrade their owncomponents). With nucleic acids, there is also considerable difficultydesorbing very high molecular weight DNA from paper matrices. Surfaceadsorption effects can cause losses of DNA and this will cause thepreferential loss of the least degraded, i.e. the most desired class ofDNA molecules. Thus the long-term archiving of biomolecules is adesirable feature of a storage medium.

Molecular and Nucleic Acid Analyses

The polymerase chain reaction (PCR) is a common tool used in molecularbiology for amplifying nucleic acids. U.S. Pat. No. 4,683,202 (Mullis,Cetus Corporation) describes a process for amplifying any desiredspecific nucleic acid sequence contained in a nucleic acid or mixturethereof.

Furthermore, U.S. Pat. No. 5,593,824 and U.S. Pat. No. 5,763,157 (Treml)describe biological reagent spheres useful for the PCR reaction.Additionally, this invention describes a convenient approach by means ofexcipient mixes comprising suitable carbohydrates useful for storage ofreagents used in downstream genetic analysis such as PCR. Carbohydratesare preferably Ficoll and melezitose. This technology has beencommercialised in a ready to go (RTG) PCR format (GE Healthcare).

Long-term storage, transport and archiving of nucleic acids on filterpaper or chemically modified matrices is a well-known technique forpreserving genetic material before the DNA or RNA is extracted andisolated in a form for use in genetic analysis such as PCR. Thus, EP1563091 (Smith et al., Whatman) relates to methods for storing nucleicacids from a sample such as cells or cell lysates. The nucleic acid isisolated and stored for extended periods of time at room temperature andhumidity, on a wide variety of filters and other types of solid phasemedia. The document describes methods for storing nucleicacid-containing samples on a wide range of solid phase matrices intubes, columns, or multiwell plates.

Cellulose derived solid supports are described by reference to thefollowing prior art.

WO 90/003959 (Burgoyne) describes a solid medium for the storage of DNA,including blood DNA, comprising a solid matrix having a compound orcomposition which protects against degradation of DNA incorporated intoor absorbed on the matrix. The document also discloses methods forstorage of DNA using this solid medium, and for recovery of DNA or insitu use of DNA or RNA.

Forensic and Human Identification Applications

DNA profiling (also called DNA testing, DNA typing, or geneticfingerprinting) is a technique employed by forensic scientists to assistin the identification of individuals by their respective DNA profiles.DNA profiles are encrypted sets of numbers that reflect a person's DNAmakeup, which can also be used as the person's identifier. DNA profilingshould not be confused with full genome sequencing. It is used in, forexample, parental testing and criminal investigations.

The method of DNA profiling is based on PCR using short tandem repeatsof nucleotide sequences. This method uses highly polymorphic regionsthat have short repeated sequences of DNA (the most common is 4 basesrepeated, but there are other lengths in use, including 3 and 5 bases).Because unrelated people almost certainly have different numbers ofrepeat units, short tandem repeats (STRs) can be used to discriminatebetween unrelated individuals. These STR loci (locations on achromosome) are targeted with sequence-specific primers and amplifiedusing PCR. The DNA fragments that result are then separated and detectedusing electrophoresis. There are two common methods of separation anddetection, capillary electrophoresis (CE) and gel electrophoresis.

Clinical Applications

A number of DNA databases created from babies' blood samples alsoexists. Blood samples taken in heel-prick tests to screen for seriousconditions are being held for years by some hospitals and can besubsequently accessed by the police to identify people involved incrimes. The samples can also be used by coroners and medical researchersfor a variety of purposes. Blood spot screening is carried out on babiesaged between five and eight days old in order to test for a variety ofserious conditions such as cancer, tumour marking and archiving, sicklecell, PKU and cystic fibrosis. Government guidelines advise hospitals tostore the samples for at least five years before destroying them.

In Denmark, for example, Danish Newborn Screening Biobank at StatensSerum Institut retains a blood sample from all neonates born after 1981.The purpose is to test for PKU and other diseases. This database is alsoused for DNA tests to identify deceased and suspected criminals.

With all the applications outlined above, however, there is a great needfor new advances for improved inert matrices that are convenient, safeand confer stability to the biomolecules which is to be analysed. Forexample, while Burgoyne (U.S. Pat. No. 5,756,126) describes a medium foranalysis of genetic material, no reference is made to other biomoleculessuch as proteins, polypeptides and metabolites.

Pathogens and Infectious Agents

Infectious diseases, also known as contagious diseases or transmissiblediseases, and including communicable diseases, comprise clinicallyevident illness (i.e., characteristic medical signs and/or symptoms ofdisease) resulting from the infection, presence and growth of pathogenicbiological agents in an individual host organism. In certain cases,infectious diseases may be asymptomatic for much or their entire course.Infectious pathogens include viruses, bacteria, bacterial spores, fungi,protozoa, and, multicellular parasites. These pathogens are the cause ofdisease epidemics, in the sense that without the pathogen, no infectiousepidemic occurs. Common examples of infectious agents includeStaphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli,Salmonella choleraesuis and Candida albicans and those that causesexually transmitted disease or septicaemia. Common microorganisms thatare routinely isolated from wounds, using clinical swabs, have includedStaphylococcus aureus, Corynebacterium sp, Candida albicans andPseudomonas aeruginosa.

Transmission of pathogen can occur in various ways including physicalcontact, contaminated food, body fluids, objects, airborne inhalation,or through vector organisms, so safe capture of infectious agent wouldbe of significant value during any diagnostic workflow. Infectiousdiseases that are especially infective are sometimes called contagiousand can be easily transmitted by contact with an ill person or theirsecretions. Infectious diseases with more specialized routes ofinfection, such as vector transmission or sexual transmission, areusually regarded as contagious. Thus inactivation of the target pathogenmay be useful prior to diagnostic testing. Sample types may include:pathological specimens from human or samples from veterinary medicinee.g. blood, urine, semen, vaginal secretions, faecal samples, CSF,tissue, lung lavage, sputum, nasopharyngeal samples, cell cultures,soil, water supplies, stream or river samples, aerial.

Once dry, samples could be transported and stored in a dark and dryenvironment. These simple conditions will ensure that the integrity ofthe biological sample on the solid medium/card is maintained. Nospecific temperature control is required for either storage ortransport, as both are carried out at room temperature.

Alginate

Alginate, a salt of alginic acid, is extracted from marine kelp(seaweed). The calcium, sodium, and ammonium alginates have been used infoam, cloth, and gauze for absorbent surgical dressings. Solublealginates, such as those of sodium, potassium, or magnesium, form aviscous sol that can be changed into a gel by a chemical reaction withcompounds such as calcium sulphate. Salts of alginic acid withmonovalent cations (Na-salt, K-salt, NH4-salt) as well as Alginate Esterare all soluble to cold and hot water, and generate viscous aqueoussolution with long-flow properties. Alginic acid and calcium alginateare water-insoluble.

Alginic: acid is substantially insoluble in water. It formswater-soluble salts with alkali metals, such as sodium., potassium, and,lithium; magnesium; ammonium; and the substituted ammonium cationsderived from lower amines such as methyl amine, ethanol amine, diethanolamine, and triethanol amine.

Alginate absorbs water quickly, which makes it useful as an additive indehydrated products, and is well known as an additive in the manufactureof paper and textiles to facilitate printing with ink and/or dyeproducts. Accordingly, paper producers are familiar with usage ofalginate components, such as sodium alginate, during manufacturing andprocessing. Sodium alginate; can make paper glossy and smooth and itraises the paper's absorption to printing ink and increases itspliability and toughness.

Alginate dressings are natural wound dressings derived from differenttypes of algae and seaweeds. These types of dressings are best used onwounds that have a large amount of exudate and may also be applied ontodry wounds after normal saline is first applied to the site ofapplication.

U.S. Pat. No. 5,820,998 (Schweitzer Maudit Int Inc.) describes a processof making a coated paper for wrapping papers used in smoking articlescomprising the steps: 1) providing a paper layer composed of a blend ofpulp fibers and particulate material containing polyvalent metalcations, 2) applying a acidified alginate solution of a materialselected from salts and derivatives of alginic acid to cover at least aportion of the paper, 3) reacting the salts and/or derivatives ofalginic acid with polyvalent metal cations in the paper to form apolymer coating, and 4) drying the paper and polymer coating. Thepermeability of the coated paper is generally at least about 75 percentless than the permeability of an identical uncoated portion of thepaper.

Bonino et al. (2011 Carbohydate Polymers 85, 111-119) describes theelectrospinning of alginate-based nano-fibres.

US 5,482,932 (Courtaulds Fibres (Holdings) Ltd) describes alginate gelswhich have the form of a fibrous paste and which particularly have analginate content (expressed as alginic acid) in the range 2 to 11percent by weight. The gels may be prepared by treating awater-insoluble or water-swellable alginate fibre, for example calciumalginate fibre, with an aqueous solution of a solubilizing salt, forexample sodium chloride. The new gels are easier to handle than knownalginate gels and are useful in wound dressing applications.

Calcium alginate is a water-insoluble, gelatinous, cream colouredsubstance that can be created through the addition of aqueous calciumchloride to aqueous sodium alginate. Calcium alginate can be used forentrapment of enzymes and forming artificial seeds in plant tissueculture. It is also incorporated into wound dressings as a homeostaticagent.

Sodium alginate is a gum, extracted from the cell walls of brown algae.As a flavourless gum, it is used by the foods industry to increaseviscosity and as an emulsifier. It is also used in indigestion tabletsand the preparation of dental impressions. Other applications includeuse in reactive dye printing, as a thickener for reactive dyestuffs, intextile screen-printing and in carpet jet-printing.

Potassium alginate is the potassium salt of alginic acid. It is anextract of seaweed and is widely used in foods as a stabilizer,thickener, and emulsifier. Its use as a pharmaceutical excipient iscurrently limited to experimental hydrogel systems. The viscosity,adhesiveness, elasticity, stiffness, and cohesiveness of potassiumalginate hydrogels have been determined and compared with values from arange of other hydrogel-forming materials.

Silver alginate is known to have antimicrobial activity. For example,some alginate wound dressings contain a silver alginate, which providesantimicrobial protection and may be considered for an infected wound.

U.S. Pat. No. 6,696,077 (Scherr) relates to the preparation ofcellulosic foam products prepared from silver alginate and derivativesthereof and process for preparing them.

U.S. Pat. No. 7,344,726 (Chitoproducts Ltd) discloses a process for thepreparation of an article having a contact biocidal property comprisinga polymer solution which contains atomic/metallic silver in suspensionor complexed with the polymer.

Percival et al (2011 Int. Wound J. 8 (3) 237-243) describes theantimicrobial efficacy of a silver alginate dressing against a broadspectrum of clinically relevant wound isolates.

Cloud et al. (2002 J Clin Microbiol. 40(10): 3838-3840) compared theperformance of various swabs and transport media routinely used tocollect specimens submitted for Bordetella culture and PCR. The authorsreported that calcium-alginate swabs inhibited the PCR and recommendedthat calcium-alginate swabs should not be used for PCR detection of B.pertussis.

Eibak et al. (2012 Anal. Chem. 84, 8783-8789) demonstrated the storageand recovery of model substances (citalopram, loperamide, methodone andsertraline) from DBS spotted sodium alginate foams using electromembraneextraction and liquid chromatography-mass spectrometry analysis. Theauthors reported that lower recoveries were obtained with the commercialcards (i.e. Whatman FTA DMPK and Agilent Bond Elut DMS) for most of themodel substances compared to the recoveries with the alginate foam.

The present invention addresses the problems associated with the roomtemperature, dry storage and subsequent analysis of biomolecules presentin samples of biological materials and provides an alternative solutionto those known or suggested by the prior art. Moreover, the inventionfurther provides a means for inactivating microbial pathogens which maybe present in the biological material.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda flat solid medium for storing at least one sample of a biologicalmaterial containing a biomolecule, the solid medium comprising a solidmatrix having sorbed thereto or incorporated therein a compositioncomprising an alginate.

It will be understood that the composition of alginate, present forexample as fibres, may be woven throughout the solid matrix.

The invention is particularly useful in genotyping, diagnostics and,predominantly, forensics applications, with amplification of low copynumber genes or low expression mRNA; short tandem repeats (STRs),alleles, loci, or other genetic materials, derived from crude biologicalsamples.

In one aspect, the solid medium additionally comprises a proteindenaturing reagent. The protein denaturing reagent can lyse cellmembranes and thus provide antimicrobial activity and/or will releasebiomolecules and cellular components onto the solid medium, therebypreventing degradation of the biological material by enzymatic activity.The protein denaturing reagent may be an ionic or anionic detergent suchas sodium dodecyl sulphate (SDS) or sodium lauryl sarcosinate.

In one preferred embodiment, the ionic detergent of the invention causesinactivation of a microorganism which has protein or lipid in its outermembranes or capsids, for example, fungi, bacteria or viruses. Thisincludes microorganisms which may be pathogenic to humans or which maycause degradation of the biomolecule.

In another aspect, the solid medium additionally comprises a freeradical trap. Examples of free radical traps include uric acid or aurate salt. Typically, free radicals are believed to be generated byspontaneous oxidation of the groups which are present, for example, indenatured serum protein of blood. Free radicals may also be generated byradiation such as UV light, x-rays and high-energy particles.

In a further aspect, the solid medium additionally comprises a chelatingagent. As used herein, a chelating agent is any compound capable ofcomplexing multivalent ions including Group II and Group III multivalentmetal ions and transition metal ions (eg., Cu, Fe, Zn, Mn, etc).According to the invention, a preferred chelating agent is a strongchelating agent such as to ethylene diamine tetraacetic acid (EDTA).Chelating agents such as a citrate or oxalate are also suitable for theinvention.

It is believed that one function of the chelating agent of the inventionis to bind multivalent ions which if present with the stored biomoleculemay partake in causing damage to the biomolecule, particularly nucleicacid. Ions which may be chelated by the chelating agent includemultivalent active metal ions, for example, magnesium and calcium, andtransition metal ions, for example, iron. Both calcium and magnesium areknown to promote nucleic acid degradation by acting as co-factors forenzymes which may destroy nucleic acid (e.g., most known nucleases). Inaddition, transition metal ions, such as iron, may readily undergooxidation and reduction and damage nucleic acids by the production offree radicals or by direct oxidation.

In one aspect, the solid medium additionally comprises a chaotrophicsalt. Guanidine salt is an example of a chaotrophic salt.

In a further aspect, the alginate is selected from the group consistingof calcium alginate, sodium alginate, potassium alginate, ammoniumalginate, magnesium alginate, lithium alginate and silver alginate.

In one aspect, the alginate has antimicrobial activity.

In a preferred aspect, the alginate is silver alginate.

Candidates for co-coating purposes which facilitate pathogeninactivation or biomolecules storage include mild detergents (e.g.Triton, SDS), chelating agents (e.g. EDTA) and uric acid or a urate saltin order to facilitate stability and to act as a free radical trap.

Paper or inert matrix could be modified directly (or simply coated) withchemical groups that will support the elution (if needed) of a specificbiomolecule. This selective elution could be based upon for example; ionexchange mechanisms (alginate as a bio-carrier will mainly rely oncharged chemical moieties).

Enzymes that hydrolyse alginates are known, which will prove useful toliberate nucleic acid, protein or other biomolecules captured on thecoated support. Alginase enzyme production from cultured Bacilluscirculans is described in Hansen et al. (1984) (Appl Environ Microbiol.47(4): 704-709).

Paper or other suitable matrices could be co-coated with alginate and/ormodified with a number of different chemicals, all of which will alterthe surface properties so that it becomes resistant to irreversiblebiomolecule absorption, thereby facilitating that elution of thebiomolecule of interest.

Additional candidates for co-coating include—

-   -   a) Self-assembled monolayer of alkanethiols.    -   b) Polyethylene glycol which has been used as a coating for        biomedical devices.    -   c) Other surface-grafted polymers that resist the adsorption of        protein include polyvinyl alcohol (PVA), polyethyloxazoline        (PEOX), poly(vinylpyrrolidone), (PVP) and poly(ethyleimine)        (PEI).    -   d) An alternative is the preparation of papers that possess a        hydrophobic surface.    -   e) A further refinement include co-coating one of the        surface-grafted polymers described above with a protein (e.g.        horse IgG, albumin) to further enhance biomolecule elution.

In another aspect, the solid matrix is selected from the groupconsisting of a cellulose matrix, a nitrocellulose matrix, acarboxymethylcellulose matrix, a polyester matrix, a polyamide matrix, apolytetrafluoroethylene matrix, a fibreglass matrix and a porous ceramicmatrix. Other solid matrices suitable for this purpose include, but arenot limited to, hydrophilic polymers including synthetic hydrophilicpolymers such as polyester and carbohydrate polymers).

In a preferred aspect, the solid matrix is a cellulose matrix.

In a particularly preferred aspect, the solid matrix is a cellulosematrix and the alginate is silver alginate.

In one aspect, the sample of biological material is selected from thegroup consisting of eukarytic cell, prokaryotic cell and prion cell. Inparticular, the sample of biological material is selected from the groupconsisting of blood, plasma, saliva, urine and buccal cells.

In another aspect, the sample of biological material contains abiomolecule selected from the group consisting of nucleic acid, protein,biopharmaceutical and polysaccharide.

In a further aspect, the solid medium additionally comprises a sample ofa biological material stored thereon.

In one aspect, the solid medium additionally comprises a sample of ananalytical process. For example, the sample is a liquid chromatographyanalysis, or an HPLC analysis.

According to a second aspect of the present invention, there is provideda method for storing a sample of a biological material containing abiomolecule on a flat solid medium comprising the steps of

-   -   a. applying a sample of a biological material containing a        biomolecule to a flat solid medium comprising alginate or        comprising a solid matrix having sorbed thereto or incorporated        therein a composition comprising an alginate; and    -   b. storing said sample of biological material on said flat solid        medium.

It will be understood that the flat solid medium of the second aspectmay consist totally of alginate or be composed of alginate fibres. Inanother embodiment, the composition of alginate, present for example asfibres, may be woven throughout the solid matrix.

In one aspect, the sample is stored on the dry solid medium withoutrefrigeration.

In another aspect, the sample is stored on the dry solid medium at atemperature in the range of 4° C. to 50° C. Typically aqueous biologicalsamples are stored at low temperatures in freezers or fridges to preventcellular degradation and microbial growth. The method of the inventionavoids the problems associated with low temperature storage andtransport, in terms of the cost of buying and running fridges andfreezers, together with the space requirements of such equipment.Transport of the samples is also facilitated since the dry solid mediacan be sent through the post without the need for any cooling.

In one aspect, the sample is stored on the solid medium for a period ofat least 1 day. Indeed the sample can be stored on the dry solid mediumfor a period selected from the group consisting of at least 1 week, atleast 1 month, at least 6 months, at least 9 months, at least 1 year, atleast 2 years, at least 5 years, at least 10 years, at least 15 yearsand at least 20 years.

According to a third aspect of the present invention, there is provideda method of analysing a sample of a biological material containing abiomolecule stored on a flat solid medium, the flat solid mediumcomprising alginate or comprising a solid matrix having sorbed theretoor incorporated therein a composition comprising an alginate, comprisingthe step of analysing the biological material for the presence and/orlevel of a biomolecule.

It will be understood that the flat solid medium of the third aspect mayconsist totally of alginate or be composed of alginate fibres. Inanother embodiment, the composition of alginate, present for example asfibres, may be woven throughout the solid matrix.

Methods for analysing the biomolecule present in the biological materialinclude for example, but are not limited to, nucleic acid amplificationby the polymerase chain reaction (PCR), short tandem repeat for humanidentification purposes and DNA profiling, isothermal amplification,additionally serological testing using antibodies, lateral flow,antibody based tests such as enzyme-linked immunosorbent assay (ELISA),Western blotting, fluorescence energy resonance transfer, massspectrometry, GC/MS and tandem mass spectrometry.

The biomolecule is selected from the group consisting of nucleic acid,protein, biopharmaceutical and polysaccharide.

In one aspect, the step of analysing the biological material for thepresence and/or level of a biomolecule is carried out directly on thesolid medium or a portion thereof. For example, the portion may be adisc which can be cut or punched from the sold support into a test tubeor well of a multi-well plate and then assayed for the presence and/orlevel of a biomolecule. This process may be carried out manually orautomatically using standard robotic laboratory equipment.

In another aspect, the method additionally comprises the step of elutingthe biological material from the solid medium prior to analysing thebiological material for the presence and/or level of the biomolecule. Itwill be understood that this process may be carried out manually orautomatically using standard robotic laboratory equipment.

In a further aspect, the solid medium additionally comprises a proteindenaturing reagent. The protein denaturing reagent can be, for example,a detergent such as a sodium dodecyl sulphate.

In yet another aspect, the solid medium additionally comprises a freeradical trap. For example, the dry solid medium may comprise uric acidor a urate salt.

In one aspect, the solid medium additionally comprises a chelatingagent. EDTA is an example of a suitable chelating agent.

In another aspect, the solid medium additionally comprises a chaotrophicsalt. An example of a suitable chaotrophic salt is a guanidine salt.

In a further aspect, the alginate is selected from the group consistingof calcium alginate, sodium alginate, potassium alginate, ammoniumalginate, magnesium alginate, lithium alginate and silver alginate.

In one aspect, the alginate has antimicrobial activity.

In a preferred aspect, the alginate is silver alginate.

In a further aspect, the solid matrix is selected from the groupconsisting of a cellulose matrix, a nitrocellulose matrix, acarboxymethylcellulose matrix, a polyester matrix, a polyamide matrix, apolytetrafluoroethylene matrix, a fibreglass matrix and a porous ceramicmatrix.

In one aspect, the solid matrix is a cellulose matrix.

In a preferred aspect, the solid matrix is a cellulose matrix and thealginate is silver alginate.

In another aspect, the sample of biological material is selected fromthe group consisting of the group consisting of eukarytic cell,prokaryotic cell and prion cell. In particular, the biological materialis selected from the group consisting of blood, saliva, plasma, urineand buccal cells.

In a further aspect, the sample of biological material contains abiomolecule selected from the group consisting of nucleic acid, protein,biopharmaceutical, polysaccharide and cellular component.

According to a fourth aspect of the present invention, there is provideda kit of parts, comprising a flat solid medium as hereinbefore describedand instructions for use.

According to a fifth aspect of the present invention, there is provideda use of a flat solid medium as hereinbefore described for collecting orstoring or analysing a sample of a biological material.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a bacterial growth inhibition test. Alginateand FTA discs exhibited zones of inhibition whereas 903 (uncoated paper)did not. Control dishes exhibited typical bacterial lawns.

FIG. 2 shows the results of an inhibition of pathogen replication test.Alginate exhibited inhibition of bacterial (Staphylococcus aureus)replication while untreated (903 paper) solid media did not.

FIGS. 3A, 3B and 3C show the data from the STR/DNA profilingexperiments. FIG. 3A shows the profile obtained from the alginate coatedmatrix; FIG. 3B shows the profile from the FTA paper; and FIG. 3C showsthe profile from the 903 paper.

FIGS. 4A, 4B and 4C show the results from experiments to detect proteinand enzyme Detection using DNase and RNase as Target Molecules. FIG. 4Ashows the detection of DNase (0.125-0.5 U) which had been added toalginate, 903 and FTA Matrices. FIG. 4B shows the detection of DNaseactivity following the addition of native DNase (0.5 U) to matriceshaving Human Embryonic Stem (10⁶) Cells Applied to the alginate, 903 andFTA matrices. FIG. 4C shows the detection of RNase activity followingthe addition of native RNase (10 μU) to matrices having Human EmbryonicStem Cells (10⁶) Cells applied to the alginate, 903 and FTA matrices.

FIG. 5 shows the results from RT PCR of β-Globin Gene Fragment fromhuman embryonic stem cells. The results are shown as agarose gel (2%)electrophoresis of the RT-amplified product in which:

-   -   Lane 1: No Template control.    -   Lane 2: RNA template isolated from the alginate matrix using RNA        spin; amplified β-globin gene fragment.    -   Lane 3: Negative control—un-extracted whole blood sample.    -   Lane 4: Negative control—un-extracted whole blood sample.    -   Lane 5: Negative control—un-extracted whole blood sample.    -   Lane 6. Internal Control Sample.    -   Lane 7: No Template Control.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms, which are used in the following description and theappended claims. Throughout the specification, exemplification ofspecific terms should be considered as non-limiting examples.

The term “flat” as used herein will mean a generally smooth, even sheet-or card-like structure which is horizontally level.

As used herein, the term “biological material” shall include anymaterial or sample originating from, derived from or obtained from abiological source. Examples will include samples of human or animalorigin, such as saliva, blood, plasma, urine and buccal cells. Otherexamples include samples from cell cultures of animal, plant, bacterial,fungal or viral origin. Yet other examples include samples containingpathogens, such as saliva, blood, plasma, urine and buccal cells.Further examples include cellular components, e.g. organelles.

The term “biomolecule” as used herein shall mean any “biomolecule” or“synthetically-derived biomolecule” as defined below:

-   -   i) A biomolecule is any organic molecule that is produced by a        living organism, including large polymeric molecules such as        proteins, polysaccharides, and nucleic acids as well as small        low molecular weight molecules such as primary metabolites,        secondary metabolites, and natural products.    -   ii) A synthetically-derived biomolecule is a “biomolecule” as        defined in i) above that is generated using recombinant DNA        technologies or chemically synthesised by other non-living        in-vitro methods.    -   iii) The term “nucleic acid” is used herein synonymously with        the term “nucleotides” and includes DNA, such as plasmid DNA and        genomic DNA; RNA, such as mRNA, tRNA, sRNA and RNAi; and protein        nucleic acid, PNA.    -   iv) A “biopharmaceutical” is a biomolecule as defined by any        of i) to iii) above which is designed or produced as a drug or        drug candidate.

As used herein, the term “sorb” means that the composition of theinvention is absorbed, adsorbed, coated or otherwise incorporated intoor onto a solid matrix in such a way as not to be readily removed fromthe matrix unless subjected to conditions which are intentionally orinadvertently performed to remove the sorbed composition from the solidmatrix.

“Alginate” as used herein is the term usually used for the salts ofalginic acid, but it can also refer to all the derivatives of alginicacid and alginic acid itself.

Herein, the term “room temperature” shall mean a temperature between 4and 50 degrees Celcius.

Materials

The Alginate Matrix (Urgosorb, silver alginate wound dressing, Lot36943, was obtained from Urgo Medical (Urgo Limited, Sullington Road,Shepshed, Loughborough, UK, LE12 9JG), 903 (W101, lot 6891711) andIndicating FTA cards (WB650060, Lot FTA6903311) were obtained from GEHealthcare (GE Healthcare Life Sciences, Amersham Place, LittleChalfont, Buckinghamshire, HP7 9NA UK).

Alternative Methods for Preparing Alginate Matrices

Other methods, which are well known in the art, can be used to preparesolid supports according to the present invention.

For example, to 10 ml of ethyl alcohol is added with stirring, 2 g ofsodium alginate and 1 g of sodium hypochlorite. 10 ml of deionised wateris added and the resultant mixture incubated at room temperature for 24hours. A solid support is immersed into the liquid containing sodiumalginate and quickly immersed in a solution of aqueous 5% (w/v) calciumchloride which is used to convert the sodium alginate to calciumalginate. The solid support is removed, excess liquid squeezed off andthe solid support washed with distilled water. After washing, the solidsupport is dried by passing over heated rollers. The dried coatedmaterial is mechanically softened (micrexed) to produce a plaintmaterial. The calcium alginate forms a hydrogel. The amount of alginateadded to the solid support can vary from between 2-100% (w/v).

Alternatively solution sodium alginate/calcium alginate may be added tothe solid support directly in the presence of glycerine as a wettingagent and ethyl alcohol to prevent a gel formation. The coated materialis dried as above.

To prepare a matrix comprising silver alginate, calcium alginate isprepared using a mixture containing for example silver nitrate, silverproteinate, silver sulfadiazine, or silver acetate, and this mixture isadded to the solid support as describe above.

Zone of Inhibition Testing

Disk diffusion antibiotic sensitivity testing is a test which usesantimicrobial-impregnated discs to test whether particular bacteria aresusceptible to a specific agent. Known quantities of bacteria are grownon agar plates in the presence of discs containing relevantantimicrobial agent. If the bacteria are susceptible to a particularantimicrobial, an area of clearing surrounds the disc where bacteria arenot capable of growing (called a zone of inhibition). This along withthe rate of diffusion of the antimicrobial agent is used to estimate thebacteria's sensitivity to the particular antimicrobial agent. Ingeneral, larger zones correlate with smaller minimum inhibitoryconcentration (MIC) of that bacterium.

Staphylococcus aureus (ATCC 25923, lot 57941605) were cultured intryptone soy agar in sterile petri dishes overnight at 37° C. 6 mm discsof alginate coated inert matrix (FTA GE Healthcare) and 903 (GEHealthcare) were applied to the agar surface of the cultures and thecultures again incubated overnight at 35° C. Each bacterial lawn wasexamined for growth inhibition (zone of inhibition) around each disc.Control cultures consisted of bacterial lawn without the addition of 6mm disc.

The results from the Zone of Inhibition experiments are shown in FIG. 1.Alginate and FTA discs exhibited zones of inhibition, 903 (uncoatedpaper) did show any inhibition of bacterial growth. Control dishesexhibited typical bacterial lawns.

This experiment shows the inhibitory nature of the alginate coatedmatrix to bacterial growth.

Inhibition of Pathogen Replication

Antimicrobial testing was also carried out using the followingprocedure. Staphylococcus aureus (ATCC 25923, lot 57941605) was culturedin Tryptone Soy Broth overnight at 35° C. without shaking. The followingday, 100 μl of the neat culture was added to Butterfields Buffercontaining 6 mm punches of alginate coated matrix, or 903 paper. Theneat cultures were mixed to disrupt the matrices. A dilution series oforganism was constructed from 10⁻⁴-10⁻⁷ cfu in Butterfields Buffer andeach sample was allowed to incubate at ambient temperature for 10minutes. 0.5 ml volumes of each sample were then plated on to TryptoneSoy Agar and the plates were allowed to dry for 10 minutes at ambienttemperature. The plates were inverted and cultured overnight at 35° C.Colony counts were carried out the following day and results fromalginate matrix were compared with control plates (uncoated 903 paper).

The results from the pathogen Inactivation experiments are shown in FIG.2. Alginate exhibited inhibition of pathogen growth, 903 (uncoatedpaper) did not show any inhibition of bacterial replication. Controldishes exhibited typical bacterial lawns.

This experiment shows the inhibitory nature of the alginate coatedmatrix to bacterial growth.

Short Tandem Repeat (STR) Profiling of Amplified DNA Sequences

This experiment was carried out to amplify DNA directly for the alginatematrix and to compare results with established matrices such as FTA and903 (Controls). DNA profiling is described here and is based on PCRwhich uses short tandem repeats (STR), which are short repeatingsequences of base pairs of DNA. This method uses highly polymorphicregions that have short repeated sequences of DNA (the most common is 4bases repeated. Because unrelated people almost certainly have differentnumbers of repeat units, STRs can be used to discriminate betweenunrelated individuals. These STR loci (locations on a chromosome) aretargeted with sequence-specific primers and amplified using PCR. The DNAfragments that result are then separated and detected using capillaryelectrophoresis. Thus, STR loci consist of short, repetitive sequenceelements 3-7 base pairs in length. These repeats are well distributedthroughout the human genome and are a rich source of highly polymorphicmarkers, which may be detected using PCR. Alleles of STR loci aredifferentiated by the number of copies of the repeat sequence containedwithin the amplified region and are distinguished from one another usingfluorescence detection following electrophoretic separation.

Direct amplification of DNA from storage card punches was followed.Direct STR profiling was carried out on duplicate punches using aPowerPlex 21 System (Product code DC8902, Promega, Southampton, UK) over28 amplification cycles. The PowerPlex 21 System allowedco-amplification and four-colour fluorescent detection of 21 loci (20STR loci and Amelogenin), including D1S1656, D2S1338, D3S1358, D5S818,D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433,D21S11, Amelogenin, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX and vWA.The PowerPlex 21 System provide all materials necessary to amplify STRregions of human genomic DNA, including a hot-start thermostable DNApolymerase, master mix and primers and this kit was used to amplify DNAdirectly from 1.2 mm punches from 10⁶ human embryonic stem cells (GEHealthcare; cell line ref: WCB307 GEHC 28) applied to alginate coatedmatrix, FTA and 903 papers. The procedure was followed exactly asoutlined in the instruction booklet (PowerPlex 21 System, Promega,Southampton, UK).

Thermal Cycling conditions over 28 cycles were as follows:

-   -   96° C. for 1 minute, then:    -   94° C. for 10 seconds    -   59° C. for 1 minute    -   72° C. for 30 seconds for 28 cycles, then:    -   60° C. for 20 minutes    -   4° C. hold

The resulting PCR products were analysed on an ABI™ 3130×1 GeneticAnalyzer capillary electrophoresis system with GENEMAPPER™ v3.2 software(Life Technologies, Paisley, UK). The STR profiles generated frompunches were taken and sample results were compared.

The results of DNA amplification and DNA profiling from the alginatecoated matrix, FTA and 903 papers are shown in FIG. 3. Full DNA profileswere obtained from the alginate coated matrix (FIG. 3A), FTA paper (FIG.3B) and 903 paper (FIG. 3C). The results from the alginate coated matrix(FIG. 3A) indicated that DNA may be stored and amplified from thismatrix.

Protein and Enzyme Detection

Protein and enzyme testing was carried out with fully configured DNaseand RNase Contamination Kits (DNase & RNase Alert QC Systems, cataloguecodes AM1970 & AM1966, Life Technologies) according to themanufacturer's instructions.

In a first series of experiments, 0.125-0.5 U of DNase was applied toalginate coated matrix FTA and 903 paper in 10 μl volumes. DNAse andRNase activity was measured as outlined below.

In a second series of experiments, 1.2 mm punches were taken from 106human embryonic stem cells (GE Healthcare; cell line ref: WCB307 GEHC28) which had been applied to alginate coated matrix, FTA and 903 papersin 10 μl volumes as above. DNAse and RNase activity was measured asoutlined below.

In a third series of experiments, 1.2 mm punches were taken from 10⁶human embryonic stem cells (GE Healthcare; cell line ref: WCB307 GEHC28) containing either 0.5 U of DNase or 10 μU of RNase added to thesecells which had been applied to alginate coated matrix, FTA and 903papers in 10 μl volumes.

Detection of DNase activity was carried out as follows using a cleavablefluorescent-labelled DNase substrate. Each punch was ejected intoseparate wells of 96-well plates. Lyophilized DNase Alert Substrate wasdissolved in TE buffer (1 ml) and dispensed (10 μl) into the test wellsof the 96-well plate. 10X DNase Alert Buffer (10 μl) and nuclease-freewater (80 μl) was added and the test solution (100 μl) incubated for 60minutes at 37° C. The DNase Alert QC System Substrate is a modified DNAoligonucleotide that emits a pink fluorescence when cleaved by DNase.For this assay, fluorescence was measured on a Tecan Ultra(excitation/emission 535/595 nm using medium gain). Solutions containingDNase activity produced a pink fluorescence, whereas solutions withoutDNase activity did not fluoresce. Thus, higher levels of DNasecorresponded to an increase in the amount of light output. Negativecontrols consisted of nuclease-free water (80 μl) in place of sample.FIG. 4A shows that

DNAase activity can be detected and quantified in a rate dependentmanner using the alginate, 903 or FTA papers.

Detection of RNase was carried out as follows using a cleavablefluorescent-labelled RNase substrate. Each punch was ejected intoseparate wells of 96-well plates. Lyophilized RNase Alert Substrate wasdissolved in TE buffer (1 ml) and dispensed (10 μl) into the test wellsof the 96-well plate. 10X RNase Alert Buffer (10 μl) and nuclease-freewater (80 μl) was added and the test solution (100 μl) incubated for 60minutes at 37° C. The RNase Alert QC System Substrate is a modified RNAoligonucleotide that emits a green fluorescence when cleaved by RNase.For this assay, fluorescence was measured on a Tecan Ultra(excitation/emission 485/535 nm using medium gain). Solutions containingRNase produced a green fluorescence, whereas solutions without RNaseactivity did not fluoresce. Thus, higher levels of RNase corresponded toan increase in the amount of light output. Negative controls consistedof nuclease-free water (80 μl) in place of sample. FIG. 4B shows thatRNAase activity can be detected and quantifie in a rate dependent mannerusing the alginate, 903 or FTA papers.

Reverse transcription (RT) PCR of Total RNA

Reverse transcriptase (RT) is an enzyme used to generate complementaryDNA (cDNA) from an RNA template, a process termed reverse transcription.Reverse transcriptase creates single-stranded DNA from an RNA template.Reverse transcription polymerase chain reaction (RT-PCR) is one of manyvariants of PCR. This technique is commonly used in molecular biology todetect RNA expression levels. RT-PCR is used to qualitatively detectgene expression through creation of complementary DNA (cDNA) transcriptsfrom RNA. RT-PCR is used to qualitatively detect gene expression throughcreation of cDNA transcripts from RNA. The technique using of end-pointRT-PCR requiring the detection of gene expression levels by the use of afluorescent dye incorporated into an agarose gel is reported here.

10⁷ human embryonic stem cells (GE Healthcare; cell line ref: WCB307GEHC 28) were applied to alginate coated matrix. Total RNA was extractedfrom this matrix using an illustra RNAspin mini RNA isolation kit (GEHealthcare, Product Code 25-0500-70). Total RNA was prepared exactly asdescribed in the instruction booklet. Yield was 312 ng/μl. Purity at260/280 nm as measured on a NanVue spectrophotometer was 1.95.

Direct RT PCR was carried out in 96-well cluster plates using illustraReady to Go—RT PCR beads 2.0 units of Taq polymerase, M-MuLV reversetranscriptase, 1.5 mM MgCl₂, 60 mM KCl, 10 mM Tris-HCL, stabilisers and40 ng template RNA, prepared above with RT-PCR carried out in 50 μlvolumes following the two-step method outlined in the instructionbooklet.

Following addition of 200 μM dNTPs, reactions were Incubated at 42° C.,30 mins, and then followed by the addition of β-globin primer sequences.

β-globin primer sequences (Sigma Genosys) were as follows:

β-globin-exon I: Sequence (5′ ... 3′) (SEQ ID NO. 1)GGT GAA CGT GGA TGA AGT TG β-globin-exon III: Sequence (5′ ... 3′)(SEQ ID NO. 2) AGC ACA CAG ACC AGC ACG T

Thermocycling conditions were as follows:

-   -   94° C., 3 min    -   94° C. 30 secs    -   55° C., 1 min    -   72° C. 30 secs    -   72° C. 5 mins    -   42 cycles    -   4° C., for ever

RN'ase free agarose gel electrophoresis (2% w/v) with 1 μl of 6X loadingbuffer, was carried out to analyse the products. Results are shown inFIG. 5. Lane 2 shows amplified β-globin gene fragment obtained fromextracted RNA from the alginate coated matrix. These data shows that itis possible to extract and amplify RNA from the alginate coated matrix.

While preferred illustrative embodiments of the present invention aredescribed, one skilled in the art will appreciate that the presentinvention can be practised by other than the described embodiments,which are presented for the purposes of illustration only and not by wayof limitation. The present invention is limited only by the claims thatfollow.

What is claimed is:
 1. A support for storing a biological materialthereon, wherein the support comprises a matrix having sorbed thereto orincorporated therein an alginate and a protein denaturing reagent. 2.The support of claim 1, further comprising a free radical trap.
 3. Thesupport of claim 1, further comprising a chelating agent.
 4. The supportof claim 1, further comprising a chaotrophic salt.
 5. The support ofclaim 1, wherein the alginate has antimicrobial activity.
 6. The supportof claim 1, wherein the alginate is selected from the group consistingof calcium alginate, sodium alginate, potassium alginate, ammoniumalginate, magnesium alginate, lithium alginate, and silver alginate. 7.The support of claim 1, wherein the alginate is silver alginate.
 8. Thesupport of claim 1, wherein the matrix is selected from the groupconsisting of a cellulose matrix, a nitrocellulose matrix, acarboxymethylcellulose matrix, a polyester matrix, a polyamide matrix, apolytetrafluoroethylene matrix, a fibreglass matrix and a porous ceramicmatrix.
 9. The support of claim 1, wherein the matrix is a cellulosematrix.
 10. The support of claim 1, wherein the support is a solidsupport, and wherein the matrix is a solid matrix.
 11. The support ofclaim 1, wherein the biological material is stored thereon, and whereinthe biological material is selected from the group consisting of aeukaryotic cell, a prokaryotic cell, and a prion cell.
 12. The supportof claim 1, wherein the biological material is stored thereon, andwherein the biological material is selected from the group consisting ofa blood, a plasma, a saliva, a urine and a buccal cell.
 13. The supportof claim 1, wherein the biological material is stored thereon, andwherein the biological material comprises a biomolecule selected fromthe group consisting of a nucleic acid, a protein, a biopharmaceutical,and a polysaccharide.
 14. A method for storing a biological material ona support comprising: applying a biological material to a supportcomprising a matrix having sorbed thereto or incorporated therein analginate and a protein denaturing reagent, and storing the biologicalmaterial on the support.
 15. The method of claim 14, wherein thebiological material is stored on the support without refrigeration. 16.The method of claim 14, wherein the biological material is stored on thesupport at a temperature ranging from 4° C. to 50° C.
 17. The method ofclaim 14, wherein the sample is stored on the support for at least oneday.
 18. A method of analyzing a biological material comprising:analyzing a biological material stored on a support comprising a matrixhaving sorbed thereto or incorporated therein an alginate and a proteindenaturing reagent for a presence, level, or both, of a biomolecule,wherein the biological material comprises the biomolecule.
 19. Themethod of claim 18, wherein the analysis step is carried out directly onthe support or a portion thereof.
 20. The method of claim 18, furthercomprising: eluting the biological material from the support before theanalysis step.